Contact PD/PI: SZTUBA-SOLINSKA, JOANNA PAR-18-714 PROJECT SUMMARY This proposal aims to shift our understanding of the epitranscriptomic regulation of long non- coding (lnc) RNA structure and function in cellular processes. LncRNAs fulfill diverse functions through structure-mediated interactions with effector molecules. Epitranscriptomic modifications, with pseudouridine (?) being the most prevalent, have been shown to affect both lncRNAs structure and function, and thus play essential regulatory roles. Yet, the overall lower stability and limited abundance of most lncRNAs, often hampers the attempts to understand their modes of action and regulation. Viruses have long provided insights into cellular physiology as they serve as natural experiments that have taken place over millions of years of evolution. Viral genomes are very compact, thus, when they encode lncRNAs, these transcripts are certainly functional. Polyadenylated nuclear (PAN) RNA is a highly abundant and stable lncRNA expressed by Kaposi's sarcoma-associated herpesvirus (KSHV). PAN is a key player involved in regulation of every stage of viral and cellular gene expression, cell cycle, and pluripotency. We will use PAN as a model lncRNA to address whether ? modification orchestrates its cellular functions. In Aim 1: we will delineate the precise location and the transient state of the ? signatures on PAN. We will address the structural consequences of ?, and identify the structural and mechanistic determinants of its deposition. We will identify proteins that recognize PAN ? to mediate phenotypic effect of this modification. In Aim 2: we will perform comprehensive characterization of PAN interactome network, including other RNAs and protein in living cells. We will elucidate the role of ? in mediating these interactions, and address their subcellular/subnuclear localization. By using state-of-the-art RNA-centric methods, i.e. target-specific single-nucleotide resolution epitranscriptomic analysis, affinity purification techniques for interactome analysis, deep- sequencing RNA structure probing, our study will fundamentally change the understanding of lncRNA-mediated mechanisms regulating cellular processes. The information gained in this system will have important implications for other viral and non-viral lncRNAs, and will lay foundation for advances in unraveling novel therapeutic targets. This proposal will also provide a platform for active multidisciplinary learning, enhancing the research environment at Auburn University and exposing undergraduates to health-related meritorious research opportunities.